The regulation of the gene coding for preproenkephalin, the precursor of the opioid peptides methionine- and leucine-enkephalin, is under investigation in several neuronal systems. A full-length cDNA clone was derived by recombinant DNA methodology and sequenced in order to proved an ideal hybridization probe for rat ppEnk mRNA. The deduced amino acid sequence of rat ppEnk (Mr 30932) is similar to those of bovine and huma ppEnk with respect to opioid peptide sequences and processing sites. Southern blot analysis of rat genomic DNA with a probe prepared from the rat ppEnk clone is consistent with the existence of a single rat ppEnk gene. The rat ppEnk probe was also found to hybridize in a highly sensitive and specific manner with rat ppEnk mRNA on Northern blots of poly(A)+ or unselected total RNA from various regions of the rat brain. A dot-blot hybridization assay was developed and used to determine the following relative abundances of ppEnk mRNA: striatum 100, hypothalamus 11.2, pons + medulla 10.8, spinal cord 10.3, cerebellum 6.1, midbrain 5.9, frontal cortex 4.6, hippocampus 2.0, thalamus 1.6. Electroconvulsive shock treatment (1 sec/day) of rats for 10 days elicited increases of 76-79% and 0-14% in the relative abundances of ppEnk mRNA in poly(A)+ RNA of the hypothelamus and striatum, respectively. The ppEnk mRNA increase in the hypothalamus may explain the previously found 65-100% increase in the hypothalamic Met-enkephalin content elicited by electroconvulsive shock. ppEnk mRNA was detected in the NG108-15 mouse neuroblastoma x rat glioma hybrid cell line by Northern blot hybridization. The abundance was 1/650 of that of the rat striatum and was increased 3.5-fold by treatment of the cells with a glucocorticoid hormone (1 MuM dexamethasone) for 4 days. This cell line is useful homogenous neuronal system for the study of ppEnk gene expression. Large cDNA libraries have been prepared from mammalian neuronal mRNA, including cat dorsal root ganglion mRNA, for the isolation of clones containing cDNA for the precursors of tachykinin neuropeptides.